1. Volume 17, Issue 2, Pages 148-159 (February 2010)
MLL-AF9-Induced Leukemogenesis Requires Coexpression of the Wild-Type Mll Allele 
Austin T. Thiel, Peter Blessington, Tao Zou, Danielle Feather, Xinjiang Wu, Jizhou Yan, Hui Zhang, Zuguo Liu, Patricia Ernst, Gary A. Koretzky, Xianxin Hua 
Cancer Cell 
Volume 17, Issue 2, Pages (February 2010)
DOI: /j.ccr
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2. Cancer Cell 2010 17, 148-159DOI: (10.1016/j.ccr.2009.12.034)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
Menin Is Required for Both H3K4 Trimethylation and H3K79 Dimethylation at Hoxa9 in MA9-Transformed Cells
(A) Western blot for menin in control or Men1 excised MA9-transformed AT1 cells, which harbored Men1f/f;Cre-ER. The cells were treated with either control DMSO (Men1f/f) or 4-OHT (Men1Δ/Δ) to excise the floxed Men1.
(B–D) ChIP assay, with two distinct amplicons, for detecting dimethylated H3K79 (B), trimethylated H3K4 (C), and menin binding (D) at Hoxa9 in Men1f/f and Men1Δ/ΔAT1 cells. Error bars denote ± SD.
Cancer Cell  , DOI: ( /j.ccr )
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4. Figure 2
WT MLL and MA9 Are Recruited to Hoxa9 in a Menin-Dependent Manner
(A and B) AT1 cells were treated with either DMSO (Men1f/f) or 4-OHT (Men1Δ/Δ) and processed for ChIP assay with either (A) anti-MLL-C or (B) anti-AF9 C terminus antibodies.
(C) THP-1 cells were transduced with either control scrambled or MEN1 shRNA-expressing lentiviruses and were used for ChIP assay with anti-AF9 or anti-Dot1L antibodies. Error bars denote ± SD. See also Figure S1.
Cancer Cell  , DOI: ( /j.ccr )
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5. Figure 3
WT Mll Is Required for Growth of MA9-Transformed Leukemia Cells and Expression of HOXA9 and CCNA2
(A) A diagram for the structure of WT MLL, MA9 fusion protein, and shRNAs targeting various parts of MLL-C but not MA9.
(B–D) AT1 cells were transduced with either vector or MLL-C shRNA 11 retroviruses and were monitored for cell number (± SD) (B); WT Mll, Hoxa9, and cyclin A (CCNA2) mRNAs (± SD) (C); and the protein levels of MLL-C and cyclin A (D). THP-1 cells were transduced with either control scrambled shRNA lentiviruses (Scram) or MLL-C shRNAs.
(E–H) The resulting cells were monitored for change in number (± SD) (E); the mRNA levels of WT Mll, Hoxa9, and CCNA2 (± SD) (F); and the protein levels of MLL-C and cyclin A (G); MLL-N, MLL-AF9 (MA9), and menin (H). See also Figure S2.
Cancer Cell  , DOI: ( /j.ccr )
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6. Figure 4
WT Mll Excision Reduces the Number of MA9-Transformed Cells and Hox Gene Expression
(A) A diagram for the floxed Mll and the primers used to detect the intact or excised Mll.
(B) 4-OHT induced excision of the floxed Mll. Spleen cells from a Mllf/f;Cre-ER mouse were cultured with either DMSO or 4-OHT; this was followed by isolation of the genomic DNA and PCR amplification.
(C) A growth curve for MA9-transformed BM cells with either Mllf/f or MllΔ/Δ (± SD, cells seeded in triplicate).
(D) Quantification of Hoxa9 and Hoxa5 mRNAs in either Mllf/f or MllΔ/Δ MA9-transformed cells (± SD). See also Figure S3.
Cancer Cell  , DOI: ( /j.ccr )
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7. Figure 5
WT MLL Is Crucial for Maximal Methylation of Both H3K4 and H3K79 at Key Target Genes
THP-1 cells were transduced with either control scrambled shRNA or MLL-C shRNA 14 lentiviruses to knock down WT MLL and were evaluated with ChIP assay for MLL-C binding to Hoxa9 (A) and for histone H3K4m3 and H3K79m2 at HOXA9 (B) and CCNA2 (C). The specificity of the anti-H3K4m3 antibody and the anti-H3K79m2 were confirmed using specifically modified peptides and Western blot. Error bars denote ± SD. See also Figure S4.
Cancer Cell  , DOI: ( /j.ccr )
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8. Figure 6
WT MLL Knockdown Suppresses Colony Formation of MA9-Transduced BM
(A) Procedure for the colony formation assay. Bone marrow (BM) cells from a C57B6 mouse were transduced with pMSCV-MA9 retroviruses and replated in triplicate weekly in methylcellulose medium with G418. After the second plating, surviving MA9 cells were transduced with each of the MLL-C shRNAs (12 and 14) or scrambled vector.
(B) A summary of colony numbers for control or Mll shRNA-transduced BM.
(C) Representative colonies from the culture plates (scale bars represent 5 mm). Error bars denote ± SD.
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9. Figure 7 WT Mll Is Required for Colony Formation of MA9-Induced BM
(A) A flowchart for procedures of MA9-induced transformation and 4-OHT-induced Mll excision.
(B) Mll excision reduced the number of colonies from MA9-transduced BM from the Mllf/f;Cre-ER mice.
(C) 4-OHT failed to reduce colony formation of MA9 retrovirus-transduced BM from the Mllf/f mice.
(D) Genotyping with genomic DNA showed that 4-OHT induced Mll excision in MA9-transformed cells with the Mllf/f;Cre-ER genotype (lane 2) but failed to induce Mll excision in MA9-transformed BM cells with Mllf/f but without the Cre-ER transgene (lane 4).
(E) WT Mll excision failed to reduce Hoxa9/Meis1-induced BM colony formation (top). 4-OHT induced WT Mll excision in Hoxa9/Meis1-transformed BM (bottom).
(F) WT Mll excision reduced colony formation from normal BM. BM from corn oil (CO) or TAM-treated Mllf/f;Cre-ER mice was plated on methylcellulose medium, and the colony number was scored at the first plating (top). WT Mll was excised in BM from TAM-treated, but not from corn oil-fed, Mllf/f;Cre-ER mice (bottom).
(G) BM from corn oil or TAM-treated Mllf/f;Cre-ER mice was first transduced with MA9 and then plated on methylcellulose medium, and the colony number was scored at the first plating. Error bars denote ± SD.
Cancer Cell  , DOI: ( /j.ccr )
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10. Figure 8 WT Mll Is Required for MA9-Induced Leukemogenesis in Mice
(A) A diagram for MA9-induced leukemogenesis and WT Mll excision in mice. TAM feeding was done 3 weeks after BM transplantation.
(B) The total peripheral blood white blood cells (WBCs) in mice transplanted with MA9-transduced Mllf/f;Cre-ER BM were measured 5 weeks after corn oil or TAM feeding.
(C) Flow cytometry analysis of peripheral blood CD11b+ MA9-transformed donor cells from transplanted mice, 5 weeks after the mice were fed with corn oil or TAM.
(D) Flow cytometry analysis of CD45.2+ splenocytes from either terminally ill and corn oil-fed mice or TAM-fed mice to detect the percentage of c-kit high cells that were FcRγII/III+/CD34+.
(E) A summary of the percentage of c-kit high cells that were FcRγII/III+/CD34+ from the corn oil or TAM-fed mice.
(F) TAM-induced Mll excision in transplanted MA9-transduced BM in recipient mice increased the survival rate of the mice. Kaplan Meier curve for mice (C57B6/B6-SJL F1) transplanted with MA9-transduced Mllf/f;Cre-ER BM that were fed with either control corn oil (n = 11) or TAM (n = 7), 3 weeks after MA9 BM transplantation.
(G) A model for menin, WT MLL, and MA9/Dot1L tripartite complex-controlled regulation of coupled yet distinct histone methylations, gene transcription, MA9-induced LSCs, and leukemogenesis. See also Figure S5.
Cancer Cell  , DOI: ( /j.ccr )
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